Increasing evidence points to the engagement of the lateral habenula (LHb) in the selection of appropriate behavioral responses in aversive situations. However, very few data have been gathered with respect to its role in fear memory formation, especially in learning paradigms in which brain areas involved in cognitive processes like the hippocampus (HPC) and the medial prefrontal cortex (mPFC) are required. A paradigm of this sort is trace fear conditioning, in which an aversive event is preceded by a discrete stimulus, generally a tone, but without the close temporal contiguity allowing for their association based on amygdala-dependent information processing. In a first experiment, we analyzed cellular activations (c-Fos expression) induced by trace fear conditioning in subregions of the habenular complex, HPC, mPFC and amygdala using a factorial analysis to unravel functional networks through correlational analysis of data. This analysis suggested that distinct LHb subregions engaged in different aspects of conditioning, e.g. associative processes and onset of fear responses. In a second experiment, we performed chemogenetic LHb inactivation during the conditioning phase of the trace fear conditioning paradigm and subsequently assessed contextual and tone fear memories. Whereas LHb inactivation did not modify rat's behavior during conditioning, it induced contextual memory deficits and enhanced fear to the tone. These results demonstrate the involvement of the LHb in fear memory. They further suggest that the LHb is engaged in learning about threatening environments through the selection of relevant information predictive of a danger.

Upon stress exposure, a broad network of structures comes into play in order to provide adequate responses and restore homeostasis. It has been known for decades that the main structures engaged during the stress response are the medial prefrontal cortex, the amygdala, the hippocampus, the hypothalamus, the monoaminergic systems (noradrenaline, dopamine and serotonin) and the periaqueductal gray. The lateral habenula (LHb) is an epithalamic structure directly connected to prefrontal cortical areas and to the amygdala, whereas it functionally interacts with the hippocampus. Also, it is a main modulator of monoaminergic systems. The LHb is activated upon exposure to basically all types of stressors, suggesting it is also involved in the stress response. However, it remains unknown if and how the LHb functionally interacts with the broad stress response network. In the current study we performed in rats a restraint stress procedure followed by immunohistochemical staining of the c‐Fos protein throughout the brain. Using graph theory‐based functional connectivity analyses, we confirm the principal hubs of the stress network (e.g., prefrontal cortex, amygdala and periventricular hypothalamus) and show that the LHb is engaged during stress exposure in close interaction with the medial prefrontal cortex, the lateral septum and the medial habenula. In addition, we performed DREADD‐induced LHb inactivation during the same restraint paradigm in order to explore its consequences on the stress response network. This last experiment gave contrasting results as the DREADD ligand alone, clozapine‐N‐oxide, was able to modify the network.

Upon stress exposure a broad network of structures comes into play in order to provide adequate responses and restore homeostasis. It has been known for decades that the main structures engaged during the stress response are the medial prefrontal cortex, the amygdala, the hippocampus, the hypothalamus, the monoaminergic systems (noradrenaline, dopamine, serotonin), and the periaqueductal gray. The lateral habenula (LHb) is an epithalamic structure directly connected to prefrontal cortical areas and to the amygdala, whereas it functionally interacts with the hippocampus. Also, it is a main modulator of monoaminergic systems. The LHb is activated upon exposure to basically all types of stressors, suggesting it is also involved in the stress response. However, it remains unknown if and how the LHb functionally interacts with the broad stress response network. In the current study we performed in rats a restraint stress procedure followed by immunohistochemical staining of the c-Fos protein throughout the brain. Using Graph Theory-based functional connectivity analyses, we confirm the principal hubs of the stress network (e.g. prefrontal cortex, amygdala, periventricular hypothalamus), and show that the LHb is engaged during stress exposure in close interaction with the medial prefrontal cortex, the lateral septum, and the medial habenula. In addition, we performed DREADD-induced LHb inactivation during the same restraint paradigm in order to explore its consequences on the stress response network. This last experiment gave contrasting results as the DREADD ligand alone, clozapine-N-oxide, was able to modify the network.GRAPHICAL ABSTRACTGRAPHICAL ABSTRACT TEXTIn this study, using immunohistochemical staining of the immediate early gene c-fos and graph theory-based functional correlational analyses, we aimed at unravelling the possible engagement of the lateral habenula (LHb) within the stress response network during acute stress exposure (10-min restraint) in rats. We found that the medial part of the LHb (LHbM) was preferentially engaged, and that this engagement was concomitant to this of structures such as the medial prefrontal cortex (mPFC), the insular cortex (Ins), hypothalamic (PVH) and thalamic (PVT) paraventricular nuclei, the extended amygdala, comprising the Bed nucleus of the stria terminalis (BNST) and the entire amygdala (AMG), as well as the dopaminergic ventral tegmental area (VTA) and the serotonergic dorsal raphe nucleus (RD). This suggests upon stressful situations the LHbM serves as a relay of cortical, thalamic, hypothalamic and temporal information, further transmitted to midbrain monoaminergic systems to probably initiate coping strategies.